The Influence Of SiRNA-survivin And Survivin Small Inhibitor YM155on Radiation Sensitivity Of Esophageal Squamous Cancer KYSE150Cell In Vitro And Vivio

IntroductionEsophageal cancer is one of common malignant tumors of digestive tract, which is reported that85%of the patients with esophageal cancer occur in developing countries. In China, the incidence of esophageal cancer is particularly high. According to the tumor-related data published by world health organization (WHO) and China, the number of patients diagnosed as esophageal cell carcinoma (ESCC) is up to250000people annually, accounting for almost half of the world’s ESCC patients. Clinicaly, surgeryis usually the first choice for esophageal cancer. However, because there is no or mild symptoms in the early stage of esophageal cancer, most of the patients are diagnosed as advanced stage esophageal cancer, tumor primary tumors is bigger, often accompanied by local invasion and or lymph node metastasis,. This great part of patients missed the best operation time o rhave no chance of radical resection, so the need for role of radiation therapy is particularly important. The5-year survival rate of esophageal cancer is10-15%when treated by radiotherapy alone. A major reason of treatment failure is the resistance of tumor cells to X-ray, resulting in uncontrolled primary lesions or local recurrence.. The main purpose of this study is to search radiosensitive molecular markers and improve the local control rate of esophageal cancer.In recent years, with the development of molecular biology, the study about molecular markers in cancer treatment emerge in an endless stream and several kinds of molecular markers have been applied to clinical practice. It is well known that apoptosis is a key index for tumor biological behavior and it plays an important role in the development of tumors. Among those molecular markers, IAP family is an important family which acted as apoptosis inhibitor factor. Their family members possess similar structure and the common features of the protein molecules are two or three series with cysteine/histidine residues of baculovirus IAP repeat sequence (BIR) and a ring structure adjacent to carboxyl terminal.. Their similar function can inhibit the terminal effect molecules (e.g., caspase-3and caspase-7) of apoptosis signaling pathways directly, so they act as negative regulators of apoptosis. Survivin gene is also known as Birc5or apoptosis suppressor gene and it is a member of apoptosis suppressor family encoded by Birc in human body. Its physiological functions involved regulation of cell mitosis, cell apoptosis and angiogenesis in tumor, etc., which are closely related with the development of tumor. Compared with other regulatory genes, Survivin gene has the advantage that it selectively expresses in tumor tissues, which means that it expressed in almost all human malignant tumors, but seldomly or did not express in terminally differentiated tissues. Its unique structure and properties have attracted many attentions. Due to its relatively specific tissue distribution, Survivin has become an important target for cancer targeted therapy. Results of many researches showed that survivin is unfavorable factor of clinical prognosis for many tumors, including breast cancer, colon cancer, lung cancer and others.Many reports revealed that high expression of survivin is associated with poor prognosis of esophageal cancer, but there is few report about the relationshiop between survivin-siRNA and survivin small molecule inhibitors YM155with esophageal radiation sensitivity. This study aims to explore the relationship between Survivin expression and the radiosensitivity and prognosis of ESCC by analyzing the Survivin expression in tumor tissues before and after radiotherapy in87esophageal cancer patients receiving preoperative radiotherapy combined with surgery; By constructing Survivin specificity of small interfering RNA (SiRNA) expression vector, human esophagus carcinoma cell line KYSE150were tranfected and then treated with Survivin small molecule inhibitors YM155K followed by X-ray irradiation. The impact of Survivin gene on cell proliferation, apoptosis, cell cycle and cell radiosensitivity were observed and the mechanism of radiosensitizing effect induced by inhibiting Survivin gene expression was also preliminarily discussed; By caculating tumor volume change of tumor-burdened mouse before and after radiation exposure, the radiosensitizing effect of Survivin gene expression was evaluated in vivo. All of the above will provide theoretical and experimental basis for the clinical application of gene therapy combined with radiation therapy. Objective 1、To detect the protein expression levels of survivin in locally advanced esophageal squamous cell carcinoma (LDESCC) before and after chemoradiation therapy and explore the association between the survivin protein expression and ESCC prognostic significance.2、To construct Survivin specificity of small interfering RNA (SiRNA) expression vectors used for transfecting esophageal squamous cell carcinoma KYSE150cell line and making itself stably expressiing and further provide a good model for the study of Survivin expression regulation function.3、To explore the impact of survivin-siRNA and survivin small molecule inhibitors on the biological behavior of KYSE150cell line and further study their influence on radiosensitivity.4、To build nude mice transplantation tumor model of esophagus carcinoma KYSE150cells and explore the effect of survivin siRNA and survivin small molecule inhibitors on cell proliferation in the tumor-bearing mouse.Methods1、A total of87patients with locally advanced disease (T3/4N0or any T stage with N1) received radiotherapy followed by surgery. The protein expression levels of survivin in biopsy samples from all the patients before and after radiotherapy were detected by the Max Envision immunohistochemistry method. The differences of survivin protein expression levels in ESCC tissue before and after radiation therapy were evaluated by Chi-square test or Chi-square test after correction. Kaplan-Meier method was used for analysis of overall survival. The Log-rank test was used for univariate prognostic analysis and Cox proportional hazards regression analyses was used for multivariable prognostic analysis.2、Packaging survivin-shRNA slow viral vector (GV118-survivin shRNA) and negative control carrier (GV118-survivin-NC shRNA) were built; Virus packaging and its titer detection were conducted in293t cells; Esophageal cancer cells KYSE150were infected by lentiviral vector and stable transfection cell lines were screened out; RT-PCR and Western-blot were performed to confirm the effectiveness of lentiviral vector.3、Based on different processing time and different concentration, the most effective group of KYSE150cells treated by survivin small molecule inhibitors (YM155) was screened out using RT-PCR and Western-blot. 4、A total of0Gy、4Gy、8Gy of6MV X-ray was irradiated to survivin-siRNA transfection group, negative transfection group, YM155treatment group and control group, respectively. RT-PCR was used to detect the expression level of survivin and caspase in4groups.5、CCK8experiment and tablet clone formation experiment were used to detect the effect of radiation (0gy,2gy,4gy,6gy,8gy,10gy) on cell proliferation and survival fraction.6、The influence of the cell cycle was observed by flow cytometry. Cell apoptosis was detected by Annexin V-PE/7-AAD double staining method.7、A stably transfected KYSE150cell lines of nude mice transplantation tumor model was constructed. The tumor volume changes were evaluated on tumor-burdened rats before and after10gy X-ray radiation, The effect of inhibition of survivin expression on radiosensitivity of esophageal tumor tissue was further explored.Statistical methodSPSS17.0statistical software was used to deal with the data. Chi-square test or adjusted chi-square test was used to testify the correlation between the expression of survivin protein with baseline characteristics of esophageal cancer and pathological response after radiotherapy. Survival rates were obtained by Kaplan-Meier method. Unvariate survival analysis was performed by Log-rank test and multivariate survival analysis was performed by Cox proportional hazards model. Data in each group were shown as x±s. One-way analysis of variance (One-way ANOVA) was used to compare the difference among group and, LSD method was used to compare the difference between each two groups. Each experiment is repeated three times and the sample size is3. P<0.05was considered as statistically significant.Results1、The positive expression rate of Survivin before and after radiotherapy of esophageal tissue were64.37%(56/87) and29.89%(26/87) respectively and its expression was significantly higher before radiotherapy than after radiotherapy in tumor tissues and the difference was statistically significant (P=0.00). Survivin protein expression in esophageal squamous carcinoma tissues before radiotherapy was not significantly correlated with the patient’s age, sex, tumor location, differentiation degree, the length of lesion and the clinical stage (P value are:0.59,0.59,0.76,0.96,0.95,0.67). Its expression after radiotherapy was also not significantly correlated with those clinical factors (P value are:0.55,0.55,0.82,0.99,0.96,0.12). Results of multivariate analysis showed that the expression of survivin protein postoperatively (chi-square=5.38, P=5.38) and pathological response postoperatively (chi-square=0.62, P=0.62) were predictors for overall survival of esophageal cancer.2、Survivin-siRNA expression vector was successfully constructed and its correctness was identified by sequencing. Results of RT-PCR showed that the expressionof survivin mRNA in transfection groupwas significantly decreased than that in negative control group and blank control group, with significant difference (P<0.05). and the inhibitory effect on survivin mRNA was the most obvious when cells were transfected after48hours.3、The inhibitory effect of Survivin small molecule inhibitors YM155on Survivin expression was both time-dependent and dose-dependent. The inhibitory rate of Survivin expression was (88.0±2.0)%when cell were treated after48hours at a concentration of100nm/L.4、After cells in each group were treated by4Gy of X-ray irradiation, G2/M phase cells in survivin transfection group and YM155processing group were significantly lower than that in negative control group and blank control group detected by flow cytometry Annexin V-PE/7-AAD double staining method, but the rate of cell apoptosis rate in survivin transfection group and YM155processing group were significantly higher than negative control and blank control group. The difference was statistically significant (P<0.05).5、Nude mouse transplantation tumor experiment confirmed that mean tumor volume reduction in Survivin expression was significantly greater than control group after3weeks of inoculation. After treated by10Gy of irradiation, the tumor volume was significantly reduced in inhibiting Survivin expression group (P<0.01).Conclusions1、Survivin protein positively expresses in esophageal squamous cell carcinoma tissues. Survivin protein expression in ESCC tissues and pathological response after radiotherapy is closely related to the prognosis of patients and they might be independent predictors of ESCC prognosis. Survivin can be used as a biological indicator screening out LAEC patients who are suitable for preoperative radiation.2、survivin siRNA and survivin small molecule inhibitors YM155have radiosensitive effect on KYSE150cells. The mechanism is related to shortening the G2/M phase cells block and improving caspase3protein expression.3、Experiments have shown that nude mouse transplantation tumor survivin gene can significantly inhibit the growth of transplanted tumor after inhibition and increase the tumor sensitivity to radiotherapy.This study suggests that Survivin might play the role of oncogene in the development of esophageal cancer. And also play a vital role in the regulation of cell proliferation and apoptosis of malignant tumors. RNA interference technology and survivin small molecule inhibitors YM155can improve radiosensitivity of esophageal cancer.